1. Field of the Invention
This invention relates to an immobilized enzyme electrode (hereinafter abbreviated enzyme electrode), and more particularly to an enzyme electrode for measuring malto-oligosaccharides, especially malto-oligosaccharides in which the degree of polymerization is different providing an equal response per wt. % concentration of the malto-oligosaccharide for each degree of polymerization and, to a measuring method using the same.
2. Description of the Prior Art
Malto-oligosaccharides are manufactured by acid hydrolysis or enzymolysis of starch. They are not only used as sweeteners or thickening agents in the food processing field, but are also widely applied as materials for pharmaceuticals or is a substrate for measuring alpha-amylase activity in the field of clinical examinations. Accordingly, the development of the method of measuring their concentration is an important issue.
Malto-oligosaccharides are usually analyzed by high performance liquid chromatography because they are water-soluble and hardly volatile compounds. In an HPLC assay, however, the pretreatment is complicated including deionization and deproteinization of the sample, and the analysis takes a long time.
Recent attention therefore has been turned to a method of analysis using an enzyme, in particular, a method using biosensors capable of analysis in a short time with almost no pretreatment and without requiring instruments such as a spectrophotometer. Among the methods of analysis using biosensors, the method most widely employed at the present time and having the highest reliability is the enzyme electrode method which is designed to oxidize or reduce the substrate enzymatically and detect the decrease of oxygen or formation of hydrogen peroxide, etc. as a current value.
At the present time, however, the enzyme capable of oxidizing or reducing the malto-oligosaccharide in one step is not practically usable. Therefore the malto-oligosaccharide is first hydrolyzed to obtain glucose, the formed glucose is then oxidized by glucose oxidase, and the decrease of oxygen or formation of hydrogen peroxide by this reaction is detected electrochemically.
On the other hand, the attempt to measure maltose by using glucoamylase and glucose oxidase is relatively old. For example, Inman et al. (D. J. Inman, W. E. Hornby: Biochem. J., 137, 25-32, 1974) immobilized glucoamylase and glucose oxidase in a nylon tube, and passed a sample containing maltose, in a continuous tube, and determined its concentration.
In the method by Inman et al. spectroscopic means were used in the final detection. Thereafter, with the purpose of detecting maltose, an enzyme electrode immobilizing glucoamylase and glucose oxidase has been studied.
The principle of an enzyme electrode method for measuring malto-oligosaccharides is as follows. In the immobilized enzyme layer on the electrode, the molecule of malto-oligosaccharide is diffused from the sample solution, and is hydrolyzed by glucoamylase as shown in the formula below, and glucose is formed. ##STR1##
In formula (1), for the sake of simplicity, the malto-oligosaccharide is expressed in an normal chain, and only a cyclic form of sugar is illustrated. Glucoamylase catalyzes the cleavage of the non-reducing terminated glucose from the malto-oligosaccharide one by one.
Glucose is further oxidized by glucose oxidase, and hydrogen peroxide is formed as shown below. ##STR2##
The generated hydrogen peroxide is oxidized by the platinum electrode, and this electrolytic current is detected as a signal. The generation rate of hydrogen peroxide is proportional to the concentration of the original malto-oligosaccharides. Thus, the concentration of the original malto-oligosaccharides is converted into a current signal and is detected.
Generally speaking, the diffusion rate of the substrate into the enzyme layer on the electrode depends on the substrate consumption rate by enzymatic reaction in the enzyme layer and the substrate concentration in the sample solution. The diffusion rate is determined so that the diffusion rate and the substrate consumption rate may be balanced. Therefore, in the enzyme layer, when the enzyme reaction rate to the unit concentration of substrate is constant, the formation rate of the enzyme reaction product formed in the enzyme layer is proportional to the substrate concentration in the sample solution.
In the clinical field, various research has concentrated on the maltose sensor for measuring alpha-amylase activity. Conventionally, in the examination of pancreatitis or hepatitis,maltopentaose was mixed with a blood sample, and the concentration of maltose in the sample was measured a specific time later. Since the maltopentaose is decomposed by alpha-amylase into, for example, maltose and maltotriose and the reducing power is increased, consequently the state of alpha-amylase activity in blood can be examined. In such an assay, when an approximate value of malto-oligosaccharide is known, the activity of alpha-amylase can be measured. It has not been attempted in the clinical field to measure accurately the malto-oligosaccharides differing in the degree of polymerization.
In actual use, however, malto-oligosaccharides do not have the same degree of polymerization of glucose, but are actually mixtures of various substances such as maltose, maltotriose and maltotetraose. In determining these malto-oligosaccharides, it is often required to measure the total glucose concentration, that is, the wt. % concentration. For example, in the fermentation process of beer or sake, the starch contained in the material is converted into alcohol through malto-oligosaccharide and glucose. In such a fermentation process, in order to measure the degree of fermentation and control the flavor of beer or sake, it is required to measure the concentration of malto-oligosaccharide as converted into glucose concentration.
However, because malto-oligosaccharides differ in the degree of polymerization, they also differ in the enzyme reaction rate by glucoamylase depending on the degree of polymerization. Therefore the apparent glucose converted concentration of malto-oligosaccharide varies depending on the composition of malto-oligosaccharides with various degrees of polymerization in the sample.
The reaction rate theory of glucoamylase is most profoundly studied among amylases. When the chain length of the malto-oligosaccharide varies, both Michaelis constant and molecular activity are changed. This is explained by the subsite theory from the viewpoint of the interaction of the enzyme active center and substrate. When malto-oligosaccharides having various chain lengths (e.g. maltose, maltotriose, maltotetraose) are hydrolyzed by glucoamylase, the rate of hydrolysis for each malto-oligosaccharide differs. When measuring by putting such enzyme and glucose oxidase into the same enzyme layer, it may be possible to determine a monodisperse oligosaccharide having only one type of chain length, but it is difficult to plot a calibration curve in a mixture. No effective analysis method has been presented so far.